Neflamapimod induces vasodilation in resistance mesenteric arteries by inhibiting p38 MAPKα and downstream Hsp27 phosphorylation

Abstract

Neflamapimod, a selective inhibitor of p38 mitogen activated protein kinase alpha (MAPKα), is under clinical investigation for its efficacy in Alzheimer's disease (AD) and dementia with Lewy Bodies (DLB). Here, we investigated if neflamapimod-mediated acute inhibition of p38 MAPKα could induce vasodilation in resistance-size rat mesenteric arteries. Our pressure myography data demonstrated that neflamapimod produced a dose-dependent vasodilation in mesenteric arteries. Our Western blotting data revealed that acute neflamapimod treatment significantly reduced the phosphorylation of p38 MAPKα and its downstream target heat-shock protein 27 (Hsp27) involved in cytoskeletal reorganization and smooth muscle contraction. Likewise, non-selective inhibition of p38 MAPK by SB203580 attenuated p38 MAPKα and Hsp27 phosphorylation, and induced vasodilation. Endothelium denudation or pharmacological inhibition of endothelium-derived vasodilators such as nitric oxide (NO) and prostacyclin (PGI₂) had no effect on such vasodilation. Neflamapimod-evoked vasorelaxation remained unaltered by the inhibition of smooth muscle cell K⁺ channels. Altogether, our data for the first time demonstrates that in resistance mesenteric arteries, neflamapimod inhibits p38 MAPKα and phosphorylation of its downstream actin-associated protein Hsp27, leading to vasodilation. This novel finding may be clinically significant and is likely to improve systemic blood pressure and cognitive deficits in AD and DLB patients for which neflamapimod is being investigated.

Figure 1

Neflamapimod stimulates vasodilation in resistance-size mesenteric arteries. (a) Original traces illustrating a concentration-dependent vasodilation in mesenteric arteries induced by acute neflamapimod (nefla) application (0.0001–10 µM) but not by the vehicle DMSO. Arrows indicate the points of diameter reading for analysis. (b) Mean data for diameter changes (over baseline) after the application of increasing concentrations of neflamapimod. (c) A sigmoidal curve illustrating concentration–response relationships between the log concentrations of neflamapimod-induced arterial vasodilation. (d) Vasorelaxation expressed as % of baseline active diameter at 80 mmHg. n = 5.

Figure 2

Neflamapimod (nefla) treatment reduces phosphorylation of p38 MAPKα in mesenteric arteries. (a) Western blot images showing the modulation of p-38 MAPKα phosphorylation but not total p-38 MAPK by nefla (10 µM), or SB203580 (10 µM) alone or together. Full-length Western blot images are presented in Suppl. Fig. 1. Mesenteric arteries were treated as indicated for 30 min and processed for Western blotting. (b) Bar graph comparing p-38 MAPKα phosphorylation normalized to GAPDH. n = 5.

Figure 3

Neflamapimod treatment reduces phosphorylation of Hsp27 in mesenteric arteries. (a) Western blot images illustrating decreases in Hsp27 phosphorylation as a function of upstream p38 MAPKα inhibition by nefla (10 µM), or SB203580 (10 µM) alone or in combination. Full-length Western blot images are presented in Suppl. Fig. 2. (b) Mean data comparing % of Hsp27 phosphorylation normalized to GAPDH. n = 5. (c) Original traces illustrating mesenteric artery vasodilation by selective and non-selective MAPKα inhibitors nefla and SB203580, respectively (d) Bar graph comparing mesenteric artery vasodilation. n = 5.

Figure 4

Role of endothelium (endo) in neflamapimod-induced vasodilation. (a) Original traces showing diameter changes by 1 µM phenylephrine (PE) alone or in the presence of 1 µM acetylcholine (ACh) or 10 µM sodium nitroprusside (SNP) in endothelium-intact and -denuded mesenteric arteries. (b) Mean data showing % reversal of PE-induced vasoconstriction by ACh and SNP, where there is a selective loss of ACh-induced vasodilation in endothelium-denuded vessels. (c) Mean data comparing 60 K-induced vasoconstriction in endothelium-intact and -denuded arteries. (d) Original traces showing that both endothelium-intact and -denuded arteries have similar magnitude of vasodilation to acute neflamapimod (10 µM) exposure. (e) Mean data for neflamapimod-evoked vasodilation in endothelium-intact and -denuded mesenteric arteries. n = 5, *P < 0.05 versus endo-intact.

Figure 5

Role of endothelial NO and PGI₂ in neflamapimod-induced vasodilation. (a) Original traces illustrating vasodilatory responses produced by neflamapimod (nefla, 10 µM) alone, and together with L-NNA (10 µM) or indomethacin (indo, 1 mM) in endothelium-intact mesenteric arteries. (b) Bar graph showing that the application of L-NNA and indo did not reduce neflamapimod-induced vasorelaxation in mesenteric arteries. n = 5.

Figure 6

Role of smooth muscle cell K⁺ channels in neflamapimod-induced vasodilation. (a) Original traces comparing vasodilation induced by neflamapimod (nefla, 10 µM) alone, and together with 4-AP (1 mM) or glibenclamide (gliben, 10 µM), or paxilline (10 µM), or TEA (tetraethylammonium, 1 mM) in endothelium-intact mesenteric arteries. (b) Mean data showing that 4-AP, gliben, paxilline or TEA did not reduce neflamapimod-induced vasorelaxation in mesenteric arteries. n = 4.